PROJECT SUMMARY During development of diverse organisms, many cells become polarized as part of the differentiation process. One example of this event occurs in the one-cell C. elegans embryo, a process that establishes the anterior-posterior (AP) body axis. In many cell types, this polarization event is cued through interactions between the actomyosin and microtubule cytoskeletons. Despite much work on polarity, the interactions between and regulation of these events are still not fully understood. PAM-1 is a puromycin-sensitive aminopeptidase required for centrosome positioning during polarity establishment in C. elegans. Puromycin-sensitive aminopeptidases (PSAs) are widely conserved and have important developmental roles in meiosis, cell cycle progression, and reproduction in organisms from plants to mice. Additionally, PSAs have been suggested to play a protective role in the development of neurodegenerative diseases such as Alzheimer?s and Huntington?s. The proposed work seeks to characterize the role of PAM-1 in regulation of the cortical actomyosin cytoskeleton. In addition, suppressors of pam-1 will be described to determine if actomyosin regulation by PAM-1 is separable from the centrosome positioning and polarity roles. In addition, the gene identities of the suppressors will be determined. Time-lapse imaging of strains bearing GFP-tagged proteins will be analyzed to follow the organization of the actomyosin cytoskeleton, the position of the centrosome, and polarity establishment in pam-1 mutants. In combination with RNAi of cytoskeletal components, the role of PAM-1 in these processes will be uncovered. Similar analysis will be done in suppressed strains to compare cytoskeletal organization, centrosome movements, and polarity in each suppressed strain. Single-nucleotide polymorphism mapping and whole genome sequencing will be employed to identify each suppressor mutation and candidates will be verified by RNAi, protein localization, and genetic analysis. It is hypothesized that cloning and characterization of these suppressor mutations will reveal novel proteins that work in conjunction with PAM-1 and as targets of the aminopeptidase during regulation of the cytoskeleton and polarity establishment. Given the implication of PSAs in neurodegeneration, identification of targets in C. elegans may also advance the understanding of disease mechanisms. Additionally, new players in centrosome positioning will be identified which may be applicable to other systems. The proposed experiments will be largely carried out by undergraduates who will be mentored to provide them the guidance and expertise needed for success in science careers and graduate work.